02 August 2013

Was Devil Facial Tumour Disease Caused By Cloned Transgenic Trees?

'It’s the only cancer ever to threaten the survival of an entire species.' Elizabeth Murchison.Download the document.SUMMARY
Much research has gone into solving the riddle of DFTD. Finding the cause of a disease can lead to it's cure. Both a cause and a cure have evaded research into DFTD for 17 years. When DFTD was first observed, research into transgenic eucalyptus trees had been under way in Tasmania for 10 years. This research used two strains of the soil bacteria agrobacterium that was used to deliver introduced genetic material into the genome of eucalyptus trees. Plant cloning was implemented both before and after genetic modification. Agrobacterium is the main cause of tumours in plants and trees and can also transfer DNA to animals and humans [1]. This paper presents a theory that a dead browsing animal such as a possum or wallaby transferred DNA from genetically modified trees to a Tasmanian devil.

THE DISEASE
First detected in 1996 by a photographer near Mount William in NE Tasmania, it soon spread among the healthy remnant population of a species that once inhabited most of Australia. The disease is new, of a very rare form and appeared to occur spontaneously. It's a stable contagious cancer that is a clone of its first manifestation. DFTD is an aggressive non-viral transmissible parasitic cancer which likely originated in Schwann cells. DFTD colonizes its devil hosts as an allogeneic graft. Allotransplantation is the transplantation of cells, tissues, or organs, to a recipient from a genetically non-identical donor of the same species.[2] The transplant is called an allograft, allogeneic transplant, or homograft. Most human tissue and organ transplants are allografts.
No immune response to the disease has been detected. This makes it different to CTVT, a transmissible cancer effecting dogs. There is only one other
recorded form of a transmissible cancer in animals and it is spread by mosquitoes. Dogs infected with CTVT develop an immune response and recover.
Viruses have been ruled out as a cause of DFTD and the mechanism by which the cells immune response is switched off is called the 'reversible epigenetic down-regulation of MHC.'[3] Somehow the devils become a living petri dish carrying a devastating oncogenic growth that developed spontaneously. No evolutionary prior stage of the disease has ever been recorded.

THE CHROMOSOMES
'The normal number of chromosomes in the devil is 14 including the XX or XY sex chromosomes. It was found that the facial tumours contained only 13 chromosomes and that these were grossly abnormal. The number and appearance of the chromosomes (the karyotype) indicated that both sex chromosomes and chromosomes 1 and 6 were absent. There was also a deletion of the long arm of chromosome 2, and four unidentified marker chromosomes were present. Most important, these anomalies were the same in the facial tumours from every animal.' [4] (corrected original work of Pearse and Swift). The full extent of chromosome changes including the complete disappearance of chromosome 1 was mapped in 2012. [5] What caused this huge re-arrangement of chromosomes almost instantly in evolutionary terms? In my view DFTD is caused by changes brought about by humans rather than by nature. Humans then 'blamed the victim' accusing devils of not having enough genetic diversity.

THE DEVELOPMENT OF TRANSGENIC EUCALYPTS TREES IN TASMANIA
The focus of GM eucalyptus research was at Ridgley South of Burnie. No official history or public disclosure has been provided concerning the Ridgley facility. The project was initiated in 1986. This report is from the Advocate newspaper in 1987 or 1988.'North Broken Hill now North Ltd acquired APPM and AFH. They established a research centre and forest nursery at Ridgley. The projects complimented each other with AFH (Australian Forest Holdings) becoming the world leaders in genetically modifying e.nitens to improve cold tolerance and pulp fibre yield.' At this time North Ltd wanted to build a large pulp mill at Wesley Vale in Tasmania, so the focus of the research was on improving pulp wood yields. The forestry subsidiary of North Ltd was renamed North Forest Products. AFH became the research and development arm of North Forest Products and was renamed North Eucalypt Technologies. Rio Tinto acquired North Limited and it's subsidiaries in 2000. They sold North Forest Products and North Eucalypt Technologies to Gunns Ltd in 2001. Other organisations involved at Ridgley were CSIRO, UTAS and ANM. High profile researcher Liz Dennis states in her bio'developed genetically engineered eucalypts for plantations which was supported by a GIRD grant in collaboration with APM, ANM, North Eucalypt Technologies, Kimberly Clarke and CSIRO Forestry.' North Forest Products also funded the GM eucalypt project.
In a series of questions on notice in the Senate seeking information on GM research Bob Brown was told: (Hansard Senate November 9 2000)
UTAS is facilitating root induction of GM nitens, globulous using agrobacterium
rhizogenes (rol genes) and agrobacterium tumefaciens (auxin biosynthesis genes) . The fact they were using auxin biosynthesis can be traced to a paper from 1986 called: 'The Hypervirulence of Agrobacterium tumefaciens A281 Is Encoded in a Region of pTiBo542 Outside of T-DNA' [6] This paper defines a 'hypervirulent' strain of agrbacterium tumefaciens called EHA101 and adds a marker gene Indole-3-acetic acid (IAA). This marker gene is kanamycin resistant. The reporter gene is -glucuronidase from escherichia coli. It is likely the Ridgley research was based on this 'hypervirulent gene' and it's goal was to enlarge the cellular fibre of trees for increased pulp wood production.

'Agrobacterium tumefaciens and A. rhizogenes are the causative agents of the crown gall and hairy root diseases, respectively. The pathogenicity of both species is caused by an inter-kingdom transfer of DNA from the bacteria to wounded plant cells. This ‘transfer-DNA’ (T-DNA) contains oncogenes whose expression transforms the plant recipient cell into a rapidly dividing tumour cell. In the case of A. tumefaciens, three of these oncogenes have been shown to encode enzymes catalyzing the biosynthesis of the plant growth hormones auxin and cytokinin. Therefore, the unorganized cell division in the crown gall tumour can be largely explained by an unregulated overproduction of these plant growth regulators. In contrast, the hairy root disease is characterized by a massive growth of adventitious roots at the site of infection. Because of the similarities of the infection processes, and because A. rhizogenes and A. tumefaciens are very closely related, it has been suggested that the most important A. rhizogenes oncogenes, the so called rol genes, are also encoding proteins involved in the regulation of plant hormone metabolism. However, recent data indicate that this is not the case. Thus the rol genes have functions that most likely are different from producing mere alterations of plant hormone concentrations'.[7]
The term 'inter-kingdom transfer of DNA' in the above quote is of interest because agrobacterium can transfer genetic material between plants, animals and humans. Agrobacterium is a prime suspect in the cause of Hodgkin’s disease in humans.[8]

THE PATENTS
The Ridgley project was linked to a Shell patent filed in February 1996. The team leader at Ridgley was named as working for Shell on GM eucalyptus in a thesis by a UTAS plant scientist.[9] The international Shell patent (WO/1996205044) was named 'Agrobacterium mediated transformation of eucalyptus'.To quote from the Shell patent 'Octopine-type border sequences were used as these have been shown to promote more efficient tumour formation when used in conjunction with the hypervirulent strain EHA101'. That links the Shell patent to the paper referenced at number 6 above. The patent continues 'In the present invention, the NPTII gene may be used as the marker gene and the resistance to a phytotoxic agent conferred by that gene, for example, resistance to G-418 (also known as geneticin) or to neomycin may be used as the characteristic for selection of transformed cells or tissue. Any other DNA sequence that confers the same or similar resistance may be used as the selectable marker.' The Shell patent was followed by many international patents involving transgenic eucalyptus including two by British And American Tobacco (BAT) under the R&D name of Advanced Technologies Cambridge. One patent applied an identical genetic manipulation to bluegums.
An interesting statement in the Shell patent is this: 'Fieldtrialling is therefore necessary after genetic manipulation, rather than before Eucalyptus trees having superior properties cannot be selected on phenotype until marked changes in the Eucalyptus morphology and physiology have occurred. These changes, from a juvenile to a mature state, can take up to 3-4 years to materialise. To maximise commercial yields and improve product quality, it is desirable to establish clonal Eucalyptus plantations, that is to say, plantations of genetically identical trees. The process of clonal propagation is repeated serially until there is sufficient cloned material to enable a plantation to be established. The resulting selected transformed plant material, for example, transformed callus, regenerating shoots or regenerated shoots, may be grown into plants directly or may be propagated vegetatively, especially by micropropagation, to increase stock before being grown into plants. Resulting genetically modified plants may themselves be cloned.'
That means the GM eucalyptus developed at Ridgley must have been field trialled. Where were they field trialled and where were the trees from the Shell patent trialled? Why is the Gene Technology Regulator and Austrade unable to advise where trials took place and when? Why did the Australian Government and North Forest Products spend millions developing GM eucalyptus trees domestically if the 'clonal plantations' were never intended to be trialled?

WHY THE FIELD TRIALS OF GM TREES REMAINED SECRET
On a timeline of events that could accomplish the transfer of agrobacterium-mediated DNA to a Tasmanian devil, North Ltd is the company most likely to have established clonal GM tree plantations in the relevant time frame. North Ltd invested directly into GM trees and the board of a listed company has a responsibility to its shareholders to invest in the best interests of that company. North Ltd had formed a partnership with the Commonwealth to develop GM eucalyptus. They were also a national mining company that owned the Uranium miner ERA. The development of the ERA Jabiluka uranium mine was dependent on the patronage of a conservative Coalition government. Without the change of government in 1996, North Ltd was unable to realise it's substantial investment in the Jabiluka uranium mine. Even so, the Jabiluka mine was so controversial it led to the arrest of 500 people and an 8 month blockade. North was a company aligned to conservative politics and not afraid to confront conservationists. The regulation of GM crops in Australia prior to 2001 were complied with voluntarily.'From 1987 through to 21 June 2001, the Genetic Manipulation Advisory Committee (GMAC), made up of an independent committee of scientific experts, assessed risks to human health and the environment. GMAC operated within an administrative system. The Committee’s recommendations regarding trials were only voluntarily complied with. In the absence of regulatory powers, GMAC had limited capacity for independent, legally enforceable auditing and monitoring of compliance. There was no legal basis for the imposition of penalties or other action in the event of non-compliance.' So it appears that in the 1980's and 1990's North Ltd did not legally have to notify anybody about trialling GM eucalyptus in Tasmania. Robin Gray was the Liberal Premier of Tasmania from 1982 to 1989. He famously recalled Parliament in 1989 on a North Broken Hill Ltd letterhead. From 1996 until his retirement on 5 May 2010, Gray was a director of forestry company Gunns Ltd, the same company that bought North Forest Products and inherited the GM trees IP. This timeline played out against the background of a polarised community, the birth of the Greens Party and divided public opinion regarding development, forestry and the natural environment. If field trials took place in Tasmania then North Ltd was the company most likely to have done it and they were kept secret.

THE SITUATION NOW
Evidence abounds about Tasmania's GM trees if you know where to look. Some of it is in media reports like this the development of 'supertrees'. Information is hidden behind arcane R&D subsidiaries like 'North Eucalypt Technologies'. The author has been in contact with the Office of the Gene Technology Regulator who provided the Hansard answers to Dr Brown's questions. OGTR claimed not to know who owned the land where GM trees were developed in Tasmania or how the research was concluded, or even if it was concluded. AusIndustry, the present incarnation of AusTrade who funded GM research in Tasmania informed me: 'the department destroyed the documentation relating to the GIRD grant fund seven years after each funding agreement expired or was otherwise terminated.' They can direct taxpayer funds into GM research and then are unable to inform the public what happened to the money or provide any documentation on GM lifeforms that can last indefinitely? The author has been in contact with a genetic plant scientist named as 'leading' the Ridgley project and who also worked on the Shell project. He denied even knowing about Ridgley even though he was contradicted in a thesis by one of his own students. This 'lead researcher' still defends GM crops but in his own words 'do not think it a good idea to risk transgenes getting into the native genepools.' Perhaps they knew what really happened to the Tasmanian devil?

If agrobacterium is genetically linked to devil facial tumour disease a prediction can be made. The 'oncogenic potential' or tumour generating properties of the chromosome will be found on the marker genes of the DFTD genome. The deleted chromosomes and lack of sex chromosomes, which I understand are not needed by trees, confirm the tumours could have jumped from the plant kingdom to devils with a dead browsing animal as an intermediary. It is known that the terpene producing properties of the trees was genetically enhanced. It would only require a single poisoning of a possum or wallaby, killed by plant terpenes or some other transgenic property of the trees, to infect devils with T-DNA bacterium in the same way plants are infected - through cellular damage. The two significant strains of DFTD may be caused by two separate poisoning events.

I would like to thank a few close friends whose relationship with the natural world as farmers and veterinarians encouraged me to develop this theory. Data-mining and data-matching were used rather than plant or animal biology. The link between agrobacterium and DFTD is a theory but as such it should remain on the table until it is scientifically disproved. The statement 'no trials of genetically modified trees ever took place in Tasmania' should not be taken at face value and requires complete documentation before it can be accepted as fact. Comments and further information are actively sought. karl@uniquecomputers.com.au

REFERENCES
[1] Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool by Stanton B. Gelvin available here.
[2] Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer by Elizabeth P. Murchison et al available here.
[3] Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer by Hannah V. Siddle et al available here.
[4] Allograft theory:Transmission of devil facial-tumour disease by A.M. Pearse and K. Swift available here.
[5] Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of a Transmissible Tumour by Janine E. Deakin et al. available here
[6] The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA by Hood et al available here.
[7] Getting to the root: The role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots by Ove Nilsson and Olof Olsson available here.
[8] Intracellular bacteria in Hodgkin’s disease and sclerosing mediastinal B-cell lymphoma:
sign of a bacterial aetiology? by Christian Sautera and Michael O. Kurrerb available here.
[9] Quantitative genetics of Eucalyptus.globulus, E.nitens and their F1 hybrid by Peter Volker available here.